Abstract : Immobilization of a low amount of electric eel acetylcholinesterase on microtiter plates by bioencapsulation in a sol-gel composite led to the preparation of a simple colorimetric enzymatic-assay for pesticides detection in olive oil. This bioassay has been used to carry out inhibition studies with three pesticides mainly used in the treatment of olive trees: Malaoxon, Methidathion-oxon and Omethoate, and tested using standard solutions and real samples of olive oil. The developed bio-tools showed a good reproducibility and stability as well as good analytical performances with limits of detection at least 10 times less than the maximum residue limit tolerated by international regulations. The obtained LODs were as low as 10-9 M for the widely used pesticide Malaoxon, 10-8 M for Methidathion oxon and 10-7 M for Omethoate. The developed test was designed for fast detection of the three target insecticides in such a complex matrix as olive oil without a laborious pre-treatment and after a simple liquid-liquid extraction, which demonstrate the potentiality of this technique to be used for commercial purpose.